Multiple Myeloma Tumor Cells Are Selectively Killed By Pharmacologically-Dosed Ascorbic Acid

Background: High-dose chemotherapy to treat multiple myeloma (MM) can be life-threatening due to toxicities to normal cells and there is a need to target tumor cells more selectively and/or lower standard drug dosage without losing efficacy. We show that pharmacologically-dosed ascorbic acid (PAA) only in the presence of high intracellular iron leads to the formation of reactive oxygen species (ROS) resulting in cell death.

Materials and Methods: CD138⁺ primary MM cells and CD138⁻ non-MM cells were isolated from 9 MM, 2 smoldering MM, and 2 monoclonal gammopathy of undetermined significance (MGUS) patients. High doses of ascorbic acid alone and combination with standard therapeutic drugs were used to treat MM cells in vitro and in vivo . Electron microscope was used to detect cell necrosis and apoptosis as well as apoptosis-inducing factor 1 (AIF1) nuclear translocation. Overexpression (OE), empty vector (EV) and knockdown (KD) of ferroportin 1 (Fpn1) and AIF1 in MM cells were achieved by lentivirus infection. An In Vivo Imaging System (IVIS) was used to monitor tumor growth in vivo .

Results: We show that PAA selectively kills CD138⁺ primary MM cells derived from MM and SMM, but not from MGUS patients. To confirm the capacity of PAA to induce MM cell death in vivo, xenografted MM mice were treated with PAA alone or combination with carfizomib or melphalan. We observed that PAA inhibited MM cell growth and extended MM mouse survival significantly. Importantly, PAA allowed a dramatic decrease in DNA-alkylating agent melphalan dosage (75%) without loss of efficacy. DFO, an iron chelator, abolished the ability of PAA to reduce cells viability in both EV and OE-Fpn1 OCI-MY5 cells pre-treated with iron. These results suggest that the mechanism of PAA killing of MM cells is indeed iron-dependent. Our data also show that PAA increases AIF1 cleavage and translocation from mitochondria to cytoplasm and nucleus. Overexpression of AIF1 in MM cells increases PAA sensitivity while KD of AIF1 prevents PAA induced MM cell death, indicating AIF1 plays an essential role in mediating PAA-induced MM cell death.

Conclusion: Our findings show that PAA only kills tumor cells with high iron content, suggesting that iron is the initiator of PAA cytotoxicity. In addition, combination of PAA with standard therapeutic drugs, such as melphalan, might significantly reduce the dose of melphalan required. Because of the elevated intracellular iron in MM cells, PAA produces highly ROS leading to release of AIF1 from mitochondria of tumor cells with no toxicity to non-tumor cells with low iron content. These results provide a mechanistic rationale to pursue the therapeutic use of PAA in MM and other cancers with high iron content. This is first study testing directly PAA efficacy on primary cancer cells in vitro .